Non-ATP-Mimetic Organometallic Protein Kinase Inhibitor.
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ABSTRACT:
SUBMITTER: Wahler K
PROVIDER: S-EPMC3892196 | biostudies-literature | 2013 Dec
REPOSITORIES: biostudies-literature
Publications
Non-ATP-Mimetic Organometallic Protein Kinase Inhibitor.
Wähler Kathrin K Kräling Katja K Steuber Holger H Meggers Eric E
ChemistryOpen 20130905 5-6
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Project description:PKG1α is a central node in cGMP signaling. Current therapeutics that look to activate this pathway rely on elevation of cGMP levels and subsequent activation of PKG1α. Direct activation of PKG1α could potentially drive additional efficacy without associated side effects of blanket cGMP elevation. We undertook a high-throughput screen to identify novel activators. After triaging through numerous false positive hits, attributed to compound mediated oxidation and activation of PKG1α, a piperidine series of compounds was validated. The hit 1 was a weak activator with EC50 = 47 μM. The activity could be improved to single digit micromolar, as seen in compounds 21 and 25 (7.0 and 3.7 μM, respectively). Several compounds were tested in a pVASP cell-based assay, and for compounds with moderate permeability, good agreement was observed between the biochemical and functional assays. These compounds will function as efficient tools to further interrogate PKG1α biology.
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Project description:Targeting sites that modulate protein-protein interactions represents an ongoing challenge for drug discovery. We have devised an assay principle, named ligand-regulated competition (LiReC), in an effort to find non-ATP competitive small-molecule regulators for type Ialpha cAMP-dependent Protein kinase (PKA-Ialpha), a protein complex that is implicated in disease. Our assay based on the LiReC principle utilizes a competitive fluorescent peptide probe to assess the integrity of the PKA-Ialpha complex upon introduction of an allosteric ligand. The developed fluorescence polarization method screens for small molecules that specifically protect (antagonists) or conversely activate (agonists) this protein complex. In high-throughput format, various cyclic nucleotide-derived agonists and antagonists are successfully detected with high precision. Furthermore, assay performance (Z'-factors above 0.7) far exceeds the minimum requirement for small-molecule screening. To identify compounds that operate through novel modes of action, our method shields the ATP-binding site and purposely excludes ATP-competitive ligands. These proof-of-principle experiments highlight the potential of the LiReC technique and suggest its application to other protein complexes, thereby providing a novel approach to identify and characterize modulators (small molecules, proteins, peptides, or nucleic acids) of protein-protein systems.
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Project description:Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) is a key regulator of the cannonical NF-κB pathway. IKKβ has been validated as a drug target for pathological conditions, which include chronic inflammatory diseases and cancer. Pharmacological studies revealed that chronic administration of ATP-competitive IKKβ inhibitors resulted in unexpected toxicity. We previously reported the discovery of 13-197 as a non-toxic IKKβ inhibitor that reduced tumor growth. Here, we show that 13-197 inhibits IKKβ in a ATP non-competitive manner and an allosteric pocket at the interface of the kinase and ubiquitin like domains was identified as the potential binding site.
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Project description:Successful engineering of functional salivary glands necessitates the creation of cell-instructive environments for ex vivo expansion and lineage specification of primary human salivary gland stem cells (hS/PCs). Herein, basement membrane mimetic hydrogels were prepared using hyaluronic acid, cell adhesive peptides, and hyperbranched polyglycerol (HPG), with or without sulfate groups, to produce "hyperGel+" or "hyperGel", respectively. Differential scanning fluorescence experiments confirmed the ability of the sulphated HPG precursor to stabilize fibroblast growth factor 10. The hydrogels were nanoporous, cytocompatibile and cell-permissive, enabling the development of multicellular hS/PC spheroids in 14 days. Incorporation of sulfated HPG species in the hydrogel enhanced cell proliferation. Culture of hS/PCs in hyperGel+ in the presence of a Rho kinase inhibitor, Y-27632 (Y-27), led to the development of spheroids with a central lumen, increased the expression of acinar marker aquaporin-3 at the transcript level (AQP3), and decreased the expression of ductal marker keratin 7 at both the transcript (KRT7) and the protein levels (K7). Reduced expression of transforming growth factor beta (TGF-β) targets SMAD2/3 was also observed in Y27-treated cultures, suggesting attenuation of TGF-β signaling. Thus, hyperGel+ cooperates with the ROCK inhibitor to promote the development of lumened spheroids with enhanced expression of acinar markers.
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Project description:Interactions between ATP and ATP-binding proteins (ATPome) are common and are required for most cellular processes. Thus, it is clearly important to identify and quantify these interactions for understanding basic cellular mechanisms and the pathogenesis of various diseases. We used an ATP competition assay (competition between ATP and acyl-ATP probes) that enabled us to distinguish specific ATP-binding proteins from non-specific proteins (Adachi et al., 2014) [1]. As a result, we identified 539 proteins, including 178 novel ATP-binding protein candidates. We also established an ATPome selectivity profiling method for kinase inhibitors using our cataloged ATPome list. Normally only kinome selectivity is profiled in selectivity profiling of kinase inhibitors. In this data, we expand the profiled targets from the kinome to the ATPome through performance of ATPome selectivity profiling and obtained target profiles of staurosporine and (S)-crizotinib. The data accompanying the manuscript on this approach (Adachi et al., 2014) [1] have been deposited to the ProteomeXchange with identifier PXD001200.
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Project description:G-protein-coupled-receptor (GPCR) signaling is exquisitely controlled to achieve spatial and temporal specificity. The endogenous protein kinase inhibitor peptide (PKI) confines the spatial and temporal spread of the activity of protein kinase A (PKA), which integrates inputs from three major types of GPCRs. Despite its wide usage as a pharmaceutical inhibitor of PKA, it was unclear whether PKI only inhibits PKA activity. Here, the effects of PKI on 55 mouse kinases were tested in in vitro assays. We found that in addition to inhibiting PKA activity, both PKI (6-22) amide and full-length PKIα facilitated the activation of multiple isoforms of protein kinase C (PKC), albeit at much higher concentrations than necessary to inhibit PKA. Thus, our results call for appropriate interpretation of experimental results using PKI as a pharmaceutical agent. Furthermore, our study lays the foundation to explore the potential functions of PKI in regulating PKC activity and in coordinating PKC and PKA activities.
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Project description:Structure-based drug design of protein-kinase inhibitors has been facilitated by availability of an enormous number of structures in the Protein Databank (PDB), systematic analyses of which can provide insight into the factors that govern ligand-protein kinase interactions and into the conformational variability of the protein kinases. In this study, a nonredundant database containing 755 unique, curated, and annotated PDB protein kinase-inhibitor complexes (each consisting of a single protein kinase chain, a ligand, and water molecules around the ligand) was created. With this dataset, analyses were performed of protein conformational variability and interactions of ligands with 11 P-loop residues. Analysis of ligand-protein interactions included ligand atom preference, ligand-protein hydrogen bonds, and the number and position of crystallographic water molecules around important P-loop residues. Analysis of variability in the conformation of the P-loop considered backbone and side-chain dihedral angles, and solvent accessible surface area (SASA). A distorted conformation of the P-loop was observed for some of the protein kinase structures. Lower SASA was observed for the hydrophobic residue in beta1 of several members of the AGC family of protein kinases. Our systematic studies were performed amino acid-by-amino acid, which is unusual for analyses of protein kinase-inhibitor complexes.
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